Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded b-lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a comparison of the amino acid sequence of select peptide fragments of Hyp1Pf11764, by conducting searches for homology with proteins in the existing nonredundant protein database, consistently revealed motifs in common with those present in bacterial response regulators that are part of two-component signal transduction systems widely distributed in bacteria.

The aim of this thesis was to gain an understanding of the molecular and ecological basis for the biological control of Pythium by fluorescent pseudomonads. A fluorescent pseudomonad biocontrol agent (BCA), Pseudomonas fluorescens 54/96, identified as a potential candidate for commercial development, was analysed together with transposon induced mutants in a variety of assays for anti-fungal activity (Chapter 2). It was revealed that 54/96 had a fungistatic effect generated by a number of different mechanisms, which included nutrient competition and antibiosis. The synecology of this organism with Pythium was then compared to a similar organism (P. fluorescens SBW25) demonstrating a similar degree of anti-fungal activity (Chapter 3). The similarity of the population dynamics of these two strains prompted an examination of the genetic basis for the anti-fungal activity of the second strain, with the intention of comparing with 54/96 (Chapter 4). Again this revealed a multifactorial mode of action of SBW25 against Pythium. Whilst some mutants with reduced anti-fungal activity were deficient in growth on seed exudate others were unaffected, but the mechanisms appeared to be different to those utilized by 54/96. The comparison of strains was expanded to a larger collection of pseudomonad BCAs which were contrasted by a number of phenotypic and genotypic methods (Chapter 5). Various markers were identified which showed commonality within the different classes of BCA, the most useful of which was cyclopropanated fatty acids. These may prove to be a useful marker when screening for new pseudomonad BCAs. It was concluded that a greater understanding of the molecular, physiological and ecological basis of anti-fungal activity of bacterial will lead to the development of biocontrol strategies with improved efficacy.

The involvement of cyanide oxygenase in the metabolism of pyruvate and a-ketoglutarate-cyanohydrin was investigated and shown to occur indirectly by the consumption of free cyanide arising from the cyanohydrins via chemical dissociation. Thus, free cyanide remains the substrate, for which the enzyme displays a remarkably high affinity (Kmapp,4 mM). A model for cyanide utilization is therefore envisioned in which the substrate is initially detoxified by complexation to an appropriate ligand followed by enzymatic oxidation of cyanide arising at sublethal levels via chemical dissociation. Putative cyanide oxygenase in cell extracts consumed both oxygen and NADH in equimolar proportions during cyanide conversion to CO2 and NH3 and existed separately from an unknown heat-stable species responsible for the nonenzymatic cyanide-catalyzed consumption of oxygen. Evidence of cyanide inhibition and nonlinear kinetics between enzyme activity and protein concentration point to a complex mechanism of enzymatic substrate conversion.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d<SUB>2</SUB>, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Copies of author's previously published articles inserted. Bibliography :leaves 65-80 Ice nucleation active (INA) bacteria were detected in a pea field in South Australia. They were identified as strains of Pseudomonas syringae and Pseudomonas flourescens biotype 1. Some chemical agents were tested on the two ice nucleating species, as cryoprotectants.

Tn5 mutagenesis of apdA (for antibiotic production) and deletion of gacA (for global antibiotic and cyanide) resulted in the same pleiotropic phenotype in Pseudomonas fluorescens (i.e. production of an array of secondary metabolites including the antibiotics pyrrolnitrin, pyoluteorin, and 2,4-diacetylphloroglucinol as well as a tryptophan-side-chain oxidase, hydrogen cyanide, and an extracellular protease was abolished). The apdA and gacA loci were identified and cloned from the genome of Pf-5. Nucleotide sequencing of the apdA and gacA loci was used to identify the open reading frames for these genes. The deduced amino acid sequences for apdA and gacA exhibited similarity to sensor kinase (ApdA) and response regulator (GacA) proteins that comprise two-component regulatory systems. The C-terminal domain of GacA containing the putative helix-turn-helix DNA-binding motif was fused to the glutathione S-transferase protein. The glutathione S-transferase GacA C-terminal fusion protein was used in a cycle selection procedure that was designed to identify GacA binding sites from a complex pool of DNA fragments. Although a putative binding site for GacA was identified using the cycle selection procedure, the results were inconclusive due to several inconsistencies in the DNA-binding assay. The upstream region of one gene, which codes for a putative porin, was identified as a putative binding site for GacA by the cycle selection procedure. Studies initiated to determine whether gacA regulates transcription of this putative porin gene have been unsuccessful, so it remains unclear whether this gene is regulated by GacA. Also, asymptotic limits to biological control of Rhizoctonia damping-off of cotton were observed with the biological control agent P. fluorescens Pf-5. / Graduation date: 1999

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

Environmental heterogeneity has often been suggested as a general explanation for patterns of diversity at scales ranging from individuals within populations to communities within landscapes. I evaluate this proposition using laboratory experiments with two microbial species, the unicellular chlorophyte Chlamydomonas reinhardtii and the common bacterium Pseudomonas fluorescens. These experiments contrast the fate of diversity following selection in heterogeneous and homogeneous environments. Specifically, I show that (1) an individual's breadth of adaptation evolves to match the amount of environmental variation, specialists evolving in environments that remain constant through time and generalists evolving in environments that vary through time irrespective of the scale at which environmental variation occurs relative to the lifetime of an individual; (2) the maintenance of diversity in a spatially heterogeneous environment is context-dependent, diversity being more readily maintained when environmental conditions are very different and genotypes are widely divergent; (3) selection in heterogeneous environments represents a plausible mechanism for two well-known patterns of diversity at large spatial scales, namely that between species diversity and both productivity and disturbance. This thesis thus demonstrates that environmental heterogeneity is a plausible, and perhaps very general, factor responsible for the diversity of natural communities.

Understanding the origins of biological diversity is a fundamental goal of evolutionary biology. A large body of theory attributes ecological and genetic diversification to divergent natural selection for resource specialization. This thesis examines adaptive radiation in response to selection for resource specialization in microcosm populations of the asexual bacterium Pseudomonas fluorescens. The general protocol for these experiments is to introduce a clonal population of Pseudomonas into a novel environment and to allow evolution to occur through the spontaneous appearance of novel genotypes carrying beneficial mutations. Adaptation can then be quantified through direct comparisons between evolved populations and their clonal ancestors. These experiments show that resource heterogeneity generates divergent natural selection for specialization on alternative resources, irrespective of the spatial structure of the environment. Adaptive radiation is possible in sympatry because of genetic trade-offs in the ability to exploit different resources, but these trade-offs are often not the result of antagonistic pleiotropy among loci that determine fitness on alternative resources. The rate of phenotypic diversification declines during adaptive radiation, apparently because the ecological opportunities required to support specialist lineages disappear as a consequence of initial diversification. The ultimate outcome of repeated instances of adaptive radiation is the evolution of a community of ecologically equivalent specialists that share similar adaptive traits, despite differences in the underlying genetic basis of specialization in replicate radiations. Comparisons with the literature on experimental evolution in microbial populations illustrate the results of this thesis are well-supported by experiments in a wide range of microbial microcosms.